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MetaMorph Super-Resolution Software Localization-based super-resolution microscopy

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Page 1: MetaMorph Super-Resolution Software - Molecular Devices · MetaMorph Super Resolution Software 7 Specifications Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

MetaMorph Super-Resolution Software

Localization-based super-resolution microscopy

Page 2: MetaMorph Super-Resolution Software - Molecular Devices · MetaMorph Super Resolution Software 7 Specifications Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

Comparison between widefield image (left) and super-resolution images in 2D (center) and 3D (right). b-tubulin labeled with Alexa647.

www.moleculardevices.com2

KEY BENEFITS• Lateral Resolution up to 20 nm

& Axial Resolution up to 40nm

• Real-time and offline super-resolution image construction

• Automatic laser feedback and adjustments

• Supports many different localization microscopy techniques

• Single molecule tracking

• Accelerated GPU processing

• Compatible with multiple hardware platforms

Break through the diffraction limitThe MetaMorph Super-Resolution Software advances imaging by enabling a 10-fold improvement in resolution.

Our MetaMorph Microscopy and Imaging Software provides a

means to control experimental hardware, capture images, perform

localization calculations, and display the developing super-resolution

image in real time.

Page 3: MetaMorph Super-Resolution Software - Molecular Devices · MetaMorph Super Resolution Software 7 Specifications Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

MetaMorph Super Resolution Software 3

Our companyOur innovative analytical solutions for cell and protein biology enable our customers to see more, do more, know more, and to answer life’s most important questions.

Abbe’s limit

The resolution of widefield optical microscopy is limited by the diffraction of light

according to the relationship discovered by Ernst Abbe. Object details smaller than

200 nm are not readily discernible by conventional light microscopy, posing a limit

for studying many biological problems at the cellular level.

Super-resolution image

A random, sparse subset of fluorescent molecules are captured and localized

while the rest of the molecules in the sample are temporarily non fluorescent.

Then, the current group of fluorescent molecules are turned non fluorescent and a

new pool of molecules are turned fluorescent.

Repeating this process a large number of times and pooling the localizations

together allows reconstruction of super-resolution image from the individual

molecule’s coordinates.

AT MOLECULAR DEVICES, WE ENABLE OUR CUSTOMERS TO UNRAVEL THE

COMPLEXITY OF BIOLOGICAL SYSTEMS

Page 4: MetaMorph Super-Resolution Software - Molecular Devices · MetaMorph Super Resolution Software 7 Specifications Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

www.moleculardevices.com4

Optimized workflow

Real-time localizations, saving, & feedbackReal-time capabilities allow reconstruction and visualization of the super-resolution image during the acquisition. With real-time feedback, the system can automatically adjust activation laser power to compensate for a changing density of localizations while acquiring images.

The number of localizations per image and laser power with real-time adjustments. When the number of localizations is outside a baseline, the laser power automatically changes to increase or decrease the density of localizations.

Acquisition workflow for acquiring, analyzing and saving images. When your image acquisition completes, the localization analysis and storage is also complete.

Acquire FrameDetect

Localizations

Store Image

Live Super Resolution

Reconstruction

Live Statistics

Automatic Laser

Adjustment

Yes

NoMore Images

3D Analysis(Gaussian Fit)

Single Molecule Tracking

Frame number

De

tect

ed

sp

ots Im

aging

laser

Page 5: MetaMorph Super-Resolution Software - Molecular Devices · MetaMorph Super Resolution Software 7 Specifications Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

5

Integrates with your protocols and systemMicroscopy techniques

Common localization microcopy techniques using photo-switchable, photo-activatable, photo-convertible proteins, or

standard fluorescent dyes are all supported.

Hardware supported

The MetaMorph Super-Resolution Software works

with many commercially available microscopes,

laser launches, TIRF optics, and can be enabled on

previously-installed imaging systems compatible with

MetaMorph Microscopy and Imaging Software.

Widefield imageof microtubules

Super-resolution image of microtubules

Calculate diffusion coefficients to analyze motionSingle molecule trackingIn addition to being able to resolve small details

in super-resolution images, you can also track

and calculate dynamics of single molecules.

After localizing all the fluorescent molecules from

the image stack, single-molecule trajectories

are computed. Mean squared distance (MSD)

and diffusion coefficient (D) are calculated for

each trajectory.

Super-Resolution image of GluA1-mEOS2

Single GluA1-mEOS2 trajectories of molecules localized and tracked more

than 8 consecutive frames

GluA1-mEOS2 DIV30 50 frames/second

MetaMorph Super Resolution Software

Page 6: MetaMorph Super-Resolution Software - Molecular Devices · MetaMorph Super Resolution Software 7 Specifications Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

www.moleculardevices.com6

3D super-resolutionAdding an optional cylindrical lens to the emission path of the microscope allows the software to precisely compute the axial position of the localized molecules. Localizations in the images are analyzed for the amount of asymmetry.

Visualization of widefield (left), super-resolution (center), and pseudocolor 3D super-resolution images (right).

An example set of 3D calibration curves for 3D super-resolution.

Set of images of sub-resolution beads for 3D super-resolution calibration.

σ (µ

m)

Z (µm)

σX

σY

Page 7: MetaMorph Super-Resolution Software - Molecular Devices · MetaMorph Super Resolution Software 7 Specifications Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

7MetaMorph Super Resolution Software

Specifications

Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

Speed2 • 100,000/second single molecule localizations • 30,000 frames in under 5 minutes (256x256 pixels, 30-50 single molecules per frame) • 850 MB/sec to SSD RAID

Computer recommendations • Windows 7x64 SP1 • At least 32GB RAM • CUDA supported NVIDIA graphics card

Features • Wavelet filtering and Gaussian fitting• 3D localization using astigmatism• Real-time super-resolution image display at any CCD frame rate• Drift correction using fiduciary markers• Automatic activation laser adjustment throughout image collection• Variable scaling of super-resolution image• Automatic thresholding and splitting of closely spaced molecules• Single molecule localization & tracking3

• Arbitrary acquired image size• Image stack acquisition• Real time statistics on the number of localizations per frame• Text file generated for data exportation

I. Izeddin, J. Boulanger, V. Racine, C.G. Specht, A. Kechkar, D. Nair, A. Triller, D. Choquet, M. Dahan, and J.B. Sibarita, “Wavelet analysis for single molecule localization Microscopy”,

Optics Express, Vol. 20, No. 3, 2081-2095 (30 January 2012).

2. Kechkar A, Nair D, Heilemann M, Choquet D, Sibarita J-B (2013), “Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy.” PLoS ONE 8(4):

e62918. doi:10.1371/journal.pone.0062918.

3. Nair D, Hosy E, Petersen JD, Constals A, Giannone G, Choquet D, Sibarita JB (2013) Super-Resolution Imaging Reveals That AMPA Receptors Inside Synapses Are Dynamically

Organized in Nanodomains Regulated by PSD95. J Neurosci 33:13204-13224.

Images courtesy of Adel Kechkar, Deepak Nair, Rémi Galland, Daniel Choquet,

Jean-Baptiste Sibarita. Interdisciplinary Institute for Neuroscience, CNRS UMR 5297,

F-33000. Bordeaux, France.

Page 8: MetaMorph Super-Resolution Software - Molecular Devices · MetaMorph Super Resolution Software 7 Specifications Resolution1 • 20 nm lateral • 40 nm axial with 3D astigmatic lens

The trademarks used herein are the property of Molecular Devices, LLC or their respective owners. Specifications subject to change without notice. Patents: www.moleculardevices.com/productpatents FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

©2015 Molecular Devices, LLC 8/15 1971A Printed in USA

Regional Offices

USA and Canada +1.800.635.5577United Kingdom +44.118.944.8000Europe* 00800.665.32860

China (Beijing) +86.10.6410.8669China (Shanghai) +86.21.3372.1088Hong Kong +852.2248.6000

Japan (Osaka) +81.6.7174.8331Japan (Tokyo) +81.3.6362.5260South Korea +82.2.3471.9531

Brazil +55.11.3616.6607

Contact Us

Phone: 800.635.5577Web: www.moleculardevices.comEmail: [email protected]

Check our website for a current listing of worldwide distributors.

*Austria, Belgium, Denmark, Finland, France, Germany, Ireland, Netherlands, Spain, Sweden and Switzerland


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